PB2158: EXPLORING THE BIOLOGICAL ROLE OF LILRA4 IN MANTLE CELL LYMPHOMA
Authors: M. K. Blum, S. V. Hansen, O. Cédile, J. Haaber, L. Chen, M. Kassem, N. Abildgaard, M. H. Hansen, C. G. Nyvold
Journal: HemaSphere (2022)

Background: Mantle cell lymphoma (MCL) is a heterogeneous, rare, and aggressive subtype of B cell non-Hodgkin’s lymphomas, with a generally poor prognosis. In a previous study, we found the leukocyte-immunoglobulin-like receptor subfamily A member 4 (LILRA4) gene to be upregulated in 3 of 4 MCL patients at both diagnosis and relapse using RNA sequencing of sorted MCL cells from bone marrow (BM) and peripheral blood (PB). Furthermore, LILRA4 expression was investigated in diagnostic PB samples from a larger cohort of patients with MCL and chronic lymphocytic leukemia using qPCR, showing that LILRA4 was overexpressed in one-half of the patients, displaying a bimodal expression profile. LILRA4 is normally expressed in monocytes, B cells, and in plasmacytoid dendritic cells. It has proven to be involved in immunomodulating processes by binding to its only known ligand, bone marrow stromal cell antigen 2 (BST2). Aims:LILRA4 remains uncharacterized in a MCL context, and the goal of this study was thus to investigate its biological role in MCL. Methods:LILRA4 mRNA expression was investigated in diagnostic samples from 33 MCL patients (26 PB and 10 BM samples) and 10 healthy donors (PB) using qPCR. Of these, 17 MCL and donor samples have previously been published (Hansen et al., 2020). Differential expression for BM and PB was evaluated using the Mann-Whitney U test (α = 0.05). Clinical information was extracted from patient files and compared to LILRA4 expression levels. GRANTA-519, a MCL cell line, were transfected with siRNA targeting LILRA4 and investigated by RNA sequencing using cells transfected with scrambled siRNA as a negative control. Results:LILRA4 transcripts showed a bimodal expression pattern for MCL PB and BM samples with high expression in half of the cases. Interestingly, the LILRA4 expression levels were significantly higher for blood compared to bone marrow (p<0.05). The LILRA4 expression level did not correlate with the degree of MCL burden in the samples, Ki67 proliferation index, mantle cell lymphoma prognostic impact (MIPI) score, or disease progression. Transcriptome sequencing was performed on LILRA4 knockdown cells to investigate a potential biological role. A stable knockdown of ~75 % was obtained at 48 to 120 hours and was confirmed by both qPCR and RNA sequencing at 72 hours post-transfection. Gene set enrichment analysis suggested a high expression of genes related to lymphomagenesis and cell cycle regulation in the MCL cell line (3.4*10^-10≤p≤1.5*10^-5, 1.7*10^-8≤FDR≤7.48*10^-5, Hallmark gene sets). A twofold expressional reduction of macrophage migration inhibitory factor, MIF, a pro-inflammatory cytokine, was observed following LILRA4 knockdown, suggesting an immunomodulating role of LILRA4 in MCL. Summary/Conclusion: In conclusion, we confirmed the bimodal LILRA4 transcript expression pattern in MCL samples at the time of diagnosis. We also demonstrated a significantly higher expression of LILRA4 in PB compared to BM, suggesting tissue-specific expression. The preliminary results from knockdown of LILRA4 indicated a potential immunomodulating role of LILRA4 through interaction with MIF, although the specific biological function of this gene in this setting remains to be elucidated. References Hansen, M. H., Cédile, O., Blum, M. K., Hansen, S. V., Ebbesen, L. H., Bentzen, H. H. N., . . . Nyvold, C. G. (2020). Molecular characterization of sorted malignant B cells from patients clinically identified with mantle cell lymphoma. Exp Hematol, 84, 7-18.e12. doi:10.1016/j.exphem.2020.03.001